The present invention relates to instrumental chemical analysis. More particularly, the invention relates to the instrumental determination of bilirubin.
Plasma unbound or “free” bilirubin (Bf) is the fraction of bilirubin not bound to plasma albumin. Bf is thought to be the bilirubin species most closely correlated with the risk of bilirubin toxicity in jaundiced newborns. Although newborn jaundice (hyperbilirubinemia) is usually a transient, benign event in the first week of life, in rare cases the accumulated unconjugated bilirubin causes irreversible brain damage or even death; see; for example, Pediatrics, 111, 1110 (2003). Technical difficulties and conflicting reports in the medical literature have limited the routine use of Bf measurements to assess newborn jaundice. Instead, the readily available plasma total bilirubin concentration (TBC) is used, a measure that has repeatedly been shown to be inferior to Bf as an indicator of bilirubin toxicity; see, e.g., Pediatrics, 93, 50 (1994); Acta Paediatr. Jpn, 34, 642 (1992); Pediatrics, 107, 665 (2001); J. Pediatr, 737, 540 (2000).
Only one FDA approved commercial device (UB-1 Analyzer, Arrows, LTD, Osaka, Japan) using the peroxidase test is available for measuring Bf. See Clin. Chem., 20, 783 (1974); J. Med. Sci, 28, 91 (1982). The accuracy of this test is limited by the need for sample dilution (forty-two fold) which may inherently alter bilirubin-albumin binding by as much as ten-fold and attenuate the effects of weak bilirubin binding competitors, which may be important clinically. See J. Biol. Chem., 276, 29953 (2001); J. Pediatr., 739, 317 (2001). The Arrows test also uses only a single concentration of peroxidase, which may cause underestimation of Bf when bilirubin dissociation from albumin is rate limiting during the test. See J. Pediatr., 708, 295 (1985). A method for measuring Bf at minimal sample dilution and additional peroxidase concentrations is therefore needed and highly desirable.